Metabolism of exogenous auxin by Arabidopsis thaliana:: Identification of the conjugate Nα-(indol-3-ylacetyl)-glutamine and initiation of a mutant screen

The accumulation of conjugates of indole-3-acetic acid (IAA) in Arabinopsis thaliana was studied by incubating tissues with high concentrations of exogenous IAA, followed by reverse phase HPLC analysis of the extracts, Using fluorescence detection, indole-3-acetyl-aspartate, indole-3-acetyl-glutamate, and indole-3-acetyl-glucose were observed and quantitated in extracts of tissue? after 24 h incubation,sith 500 mu M IAA. In addition, a new metabolite was detected and positively identified as indole-3-acetyl-glutamine by fast atom bombardment mass spectrometry, exact mass measurement, and tandem mass spectrometry in comparison with a synthetic standard. The amounts of individual conjugates formed differed between lea ces, shoot axes and roots. In all three tissues, indole-3-acetyl-aspartate was the most abundant conjugate, the highest lec el being observed in roots, Highest levels of indole-3-acetyl-glutamine were observed in leaves, where it was the second most abundant conjugate and comprised approximately 12% of the fluorescent metabolites. Accumulation of the three amide conjugates was dramatically inhibited by cycloheximide, whereas accumulation of indole-3-acetyl-glucose was little affected. Based on these data, a screen for Arabidopsis mutants altered in the IAA-inducible system for auxin conjugate formation was initiated. The first mutant to be isolated and characterized produces more indole-3-acetyl-glutamine and less indole-3-acetyl-aspartate than wild-type, and is allelic to an existing class of photorespiration mutants (gluS) deficient in chloroplastic glutamate synthase.